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Pestivirus bovine viral diarrhea virus type 1 species is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, four hundred-eighty-two genomic sequences have been evaluated applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure alignment and computing genetic distance among strains in the 5′ untranslated region of Pestivirus RNA, the current taxonomy of the species was reviewed. Twenty-two genotypes have been identified, applying a nomenclature based on divergence in the genus.

Two-hundred and eighty-one strains of bovine viral diarrhea virus type 1 (BVDV-1) were evaluated according to the palindromic nucleotide substitutions (PNS) method, a simple and practical genotyping procedure, at the three variable loci, V1, V2 and V3, in the 5′-untranslated region of genomic RNA. Ten PNSs were characteristic to the Pestivirus genus, located in the V1 and V2 loci. The BVDV-1 species shared a U-A pairing, a species-characteristic PNS, in position fifteen common to the V1 locus, and a G-C pairing in position five common to the V2 and V3 loci. Within the BVDV-1 species, 15 genotypes, BVDV-1a, BVDV-1b, BVDV-1c, BVDV-1d, and the newly described from BVDV-1e to BVDV-1o were identified based on characteristic nucleotide base pairings. The nomenclature reflected the genotypes’ level of divergence within the species. Genotypes BVDV-1l, BVDV-1m and BVDV-1o were borderline in the species.

The genus Pestivirus from the family Flaviviridae is represented by four established species; Bovine viral diarrhea virus 1 (BVDV-1); Bovine viral diarrhea virus 2 (BVDV-2); Border disease virus (BDV); and Classical swine fever virus (CSFV); as well a tentative species from a Giraffe. The palindromic nucleotide substitutions (PNS) in the 5′ untranslated region (UTR) of Pestivirus RNA has been described as a new, simple and practical method for genotyping. New software is described, also named PNS, that was prepared specifically for this PNS genotyping procedure. Pestivirus identification using PNS was evaluated on five hundred and forty-three sequences at genus, species and genotype level using this software. The software is freely available at www.pns-software.com .

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5′-untranslated region (UTR) of the Pestivirus genome have been considered for taxonomical segregation of the species, through the evaluation of 534 strains. On the basis of qualitative and quantitative secondary structure characteristics, species have been identified within the genus, determining genetic distances between species isolates, clarifying borderline and multirelated sequences, and characterizing and clustering the Pestivirus strains showing unexpected genomic sequences. Nine genomic groups have been identified: the species Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV) and the tentative species Pronghorn, Giraffe, Bovine viral diarrhea virus 3 (BVDV-3) (HoBi group), Border disease virus 2 (BDV-2) (Italian small ruminant isolates) and Bungowannah. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low variable positions (LVP) including base-pairs present in less than 80% of the genus. The determination of divergence between single strain sequences or genetic groups was obtained easily by comparing base-pairing combinations from aligned secondary structures. This provided clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. The BVDV-1 and BDV species resulted heterogeneous, showing isolates located on a borderline in the species. Within the BVDV-2 species, two main genogroups were identified, with strains showing common sequence characteristics to both groups (multirelated strains). They could be allocated correctly by quantitative analysis. Similarly, the relation between CSFV and BDV species appeared very clearly. Also in this case, ambiguous strain sequences could be clustered in the species showing the lowest divergence values. In conclusion, the proposed taxonomical procedure is based on the evaluation of only the strategic and highly conserved genome regions in the 5′-UTR. Furthermore, the application of quantitative analytical procedure allowed for a better determination of relation among species.

Two lots of polyvalent live vaccines for human use against poliovirus were tested by reverse transcriptase (RT) and nested PCR for the presence of contaminating pestivirus RNA. By RT-PCR, samples from both lots showed a band of approximately 450 bp instead of 300 bp for the reference pestivirus strain used as positive control. After nested PCR, the template DNA (450 bp product) was not amplified, suggesting non-specificity of the previous amplification. Sequencing analysis confirmed the non-specificity of the 450 bp bands and revealed, respectively, 80 and 77% homology with a region in the VP1 gene of poliovirus type 1 in samples 1 and 2. This suggests that more caution should be taken in interpreting the results obtained by PCR, and that they should be confirmed by nested PCR or sequencing.

The HCV stem–loop subdomains III-a, -b and -c have been shown to reflect the characteristics of the virus and identify isolates by genus, genotype and subtype. The aim of this study was to investigate the genotype-specific PNS within the 5′UTR of prevalent HCV genotypes (1 and 5a) found in South Africa. The genotype 5a (N = 35) and genotype 1 sequences (N = 20) were from patients presenting with liver disease or haemophilia, respectively. PNS HCV typing characteristics, defined previously, were observed. The PNS method differentiated subtypes 1a and 1c from subtype 1b by the base change at nucleotide position 243. A lack of structural data from the variable loci V1 of the 5′UTR did not allow us to further differentiate the subtypes of 1. A nucleotide change from a thymine (T) to a cytosine (C) at position 183 was found among genotype 5a sequences. This mutation changed the stable U–AA bond to a Y AA bulge at base-pair position 32. There was an insertion of a single adenine (A) at position 207. At present PNS analysis is labour intensive but, with development of further software to aid the computer analysis, it has the potential to provide a rapid, reliable alternative to phylogenetic analysis.

The palindromic nucleotide substitutions (PNS) in the 5′-untranslated region (UTR) of Pestivirus RNA have been described as a new, simple and practical method for genotyping. Given the genetic relatedness between Pestivirus and hepatitis C virus species, the application of the method was investigated preliminarily on 180 isolates, including reference strains. The keys for hepatitis C virus identification have been determined at the genus, species, genotype and subtype levels. Secondary structure nucleotide substitutions were characteristics to the genus included in a complex stem-loop structure composed of 112–115 nucleotides. Due to the worldwide importance of hepatitis C virus, and the difficulties encountered in the control of the disease, it is, therefore, important to understand the genetic aspects of the virus. The application of the PNS method might represent an additional useful tool for determining the genetic variations among hepatitis C virus strains. The identification of viral types or subtypes based on genetic changes should improve our understanding of hepatitis C virus and might provide markers for biological differences, such as virulence, and improve understanding of the evolution of the virus.

The palindromic nucleotide substitutions (PNS) at the three variable loci (V1, V2 and V3) in the 5' untranslated region (UTR) of Pestivirus RNA have been considered for taxonomical segregation of species, through the evaluation of 430 genomic sequences. On the basis of qualitative and quantitative secondary structure characteristics, six species have been identified: Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2), Classical swine fever virus (CSFV), Border disease virus (BDV), the tentative species Giraffe and a new proposed taxon named Pronghorn. The first step was qualitative and consisted in the characterization of the different positions of the three stems and loops in the 5' UTR sequences of all the strains under consideration belonging to the genus. Secondary structure sequences showing divergent base-pair combinations have been aligned for comparison. Palindromic positions have been characterized according to changes in nucleotide base-pairs identifying low-variable positions (LVP) including base-pairs present in less than 80% of the genus. The second step was quantitative, allowing the identification of genomic groups by clustering the base-pair combinations according to LVP. Relatedness among types was evaluated to identify homogeneous groups. Cross comparisons between types within the genus have been evaluated by computing the divergence percentage thus clarifying borderline and multirelated sequences.

A simple and practical method was developed for pestivirus genotyping based on analysis of the secondary structures in the 5'-untranslated region (UTR). Three stable stem-loop structures, V1, V2 and V3, predicted by computer in the 5'-UTR, included strictly conserved consensus base-pairings which are shared by all the genotypes of pestivirus or are characteristic to each genotype of pestivirus. On the basis of the palindromic nucleotide substitution at the secondary structural level, six genotypes have been identified among pestivirus strains, irrespective of the cytopathic and non-cytopathic biotypes. They are genotypes Ia, Ib, Ic and II in bovine viral diarrhea virus, genotype III in border disease virus, and genotype IV in classical swine fever virus. The stable stem-loop structures, which were maintained by palindromic nucleotide substitutions in the stem region, may represent references for the classification and identification of pestivirus species and/or genotypes.